UNIT 8: FISH

● used to check the cause of trisomies,

microdeletion syndromes, etc.
TRANS OUTLINE

I. FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)
A. SPECIMEN TYPES FOR FISH
II. FISH PROBES
A. TYPES OF FISH PROBES
o Locus Specific Probe
o Alphoid or Centromeric Repeat
o Subtelomere Probe
o Whole Chromosome Probes
o Pre-Natal Fish Probes
What is FISH?
III. STEPS FOR FISH
● Fluorescence In-Situ Hybridization. Detects and
IV. CELL SCORING IN FISH localizes specific DNA sequences using
V. APPLICATION OF FISH fluorescently labeled complementary DNA
VI. SPECIALIZED AND EVOLVING probes.
TECHNOLOGIES ● Probe: short sequence of DNA complementary to
A. COMPARATIVE GENOMIC HYBRIDIZATION target we want to detect
(CGH) ON METAPHASE CELLS ● Denature: to separate DNA strands and allow
B. MULTIPLEX FISH (M-FISH) probe access to target DNA.
C. MULTICOLOR BANDING (mBAND) ● Hybridize: together to bind probe to target DNA.
● Analyze: probe signals using a fluorescent
ANALYSIS
microscope.
D. FIBER FISH
E. PRIMED IN SITU LABELING (PRINS)
F. REVERSE FISH SPECIMEN TYPES FOR FISH
VII. COMPARATIVE GENOMIC HYBRIDIZATION

FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) Metaphase FISH Interphase FISH

Gold Standard and May also be done on
● A cytogenetic technique that uses fluorescent routinely done. uncultured specimens.
probes that bind specifically to a part of Done on cultured cells. Advantageous in the rapid
chromosomes complementary to its sequence. screening of many nuclei
● useful in detecting and mapping the presence for prenatal diagnosis and
or absence of particular DNA sequences within newborn studies.
chromosomes. Allows direct Also beneficial in the
● FISH is applied to provide specific localization visualization of study of samples with a
of genes on chromosomes. chromosomes and low mitotic index such as
● rapid diagnosis of trisomies and exact position of most solid tumors.
microdeletions is acquired using specific signals.
probes.

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 Useful in the detection Major disadvantage is the
of structural changes in inability to detect
the genome. unknown structural
chromosomal changes.
Samples: Samples:
1. Amniocytes 1. Amniocytes - for
2. Chorionic ploidy analysis
villous cells during prenatal
3. Lymphocytes studies.
4. Cells from bone 2. Peripheral Blood
marrow Smears - for
aspirates or ploidy analysis in
solid tumors. newborns.
5. Fibroblasts. 3. Bone Marrow
Aspirate Smear or
Direct Harvest -
translocation or
copy number
analysis in cancer
studies.

FISH PROBES

● Complementary sequences of target nucleic

acids (DNA, RNA, or Nucleic Acid analogs)
tagged or labeled with fluorophores.
● Designed to hybridized with the
complementary sequence
● Size ranges from 20 to 1000 base pairs.
● Direct Labelling TYPES OF FISH PROBES
○ fluorophores are directly attached to
the probe. LOCUS SPECIFIC PROBE
○ less sensitive.
○ most common and one step only ● binds to a particular region of a chromosome.
○ ex: FITC, rhodamine, and cyanines. ● used when only a small portion of a gene is
● Indirect Labelling isolated and wants to determine on which
○ chemical conjugation of the nucleic chromosome the gene is located, or how many
acid with a nonfluorescent molecule copies of a gene exist within a particular
that can bind fluorescent material genome.
after hybridization. ● Locus = defined by DNA sequence
○ ex: Biotin and Diogoxigenin.
SINGLE COLOR FISH DOUBLE COLOR FISH
PROBE PROBE

Designed to cover a gene Designed to cover any 2

of interest. genes for the detection
of the aberrations.

Allows simultaneous

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 detection of numerical
abnormalities of 2 to 3
regions in one FISH
assay.

ALPHOID OR CENTROMERIC REPEAT

● Generated from repetitive sequences found in

the middle of each chromosome.
● Used to determine whether an individual has
the correct number of chromosomes or if there
is aneuploidy in the patient’s genome.

WHOLE CHROMOSOME PROBES

● Collection of smaller probes that bind to the

whole length of the chromosome.
● Useful in the examination of chromosomal
aberrations.
○ Find missing chromosomes

SUBTELOMERE PROBE

● Specific to the subtelomere region of the

chromosome.
● Useful in the detection of subtelomere
deletions and rearrangements.

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 PRE-NATAL FISH PROBES
CELL SCORING IN FISH
● Comprise of different combinations of
fluorophore-labeled probes specific for Single color FISH counting guidelines:
chromosomes 13, 18, 21, X, and/or Y.
PICTURES RULES

Don’t count, skip over.

This could be 2 nuclei
with 1 signal to each
other or one twisted
nucleus.

Count as 2 signals. One

is very compact, the
other is diffuse.

STEPS FOR FISH

Don’t count; skip over.
Steps: Observer cannot
1. Probe and target DNAs are denatured using determine which
high-temperature incubation in a nucleus contains the
formamide/salt solution. signals.
2. Probe sequences hybridize to the
complementary target sequences, and
nonspecific binding is eliminated via stringent Count as 2 signals. 1
washing. signal in a split.
3. The probe hybridization is detected with
fluorescence microscopy.

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 Count as 3 signals. Count as 1 red signal
and 1 green signal. The
red signal is diffuse.

Count as 3 signals. 1 is
split. APPLICATION OF FISH
1. Detection and characterization of
chromosome.

Count as 4 signals.

Two color (e.g. X and Y) counting guidelines:

PICTURES RULES
Slide 22. Example of FISH to a single copy target using a
Don’t count. Nuclei are
cosmid (SNRPN) to the Prader-Willi “critical region”
overlapping and all areas
localized to 15q11-13.
of both nuclei are not
visible.

Count as 1 red signal

and 1 green signal. The
red signal is diffuse.

Don’t count. Nuclei are

too close together to
determine boundaries.
Slide 22. Partial metaphase spread from a patient with a
duplication involving chromosome 11.

2. Detection and analysis of prenatal

chromosomal abnormalities.

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 Slide 23. Prenatal ploidy assessment utilizing Abbott Slide 25. ERBB2 (HER2) analysis for carcinoma of the
Molecular AneuVysion analysis of uncultured amniotic breast.
fluid cells using unique copy probes for the long arms of
chromosomes 13, 18, 21, X, and Y.
SPECIALIZED AND EVOLVING TECHNOLOGIES
3. Study of chromosomal abnormalities
associated with cancer. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) ON
METAPHASE CELLS

● A technique that uses DNA from the cells of

interest, rather than using a standard
karyotype, for chromosomal analysis.
● This can be very useful, especially in some
cancers when only DNA is available rather than
any growing cells.
● This technology has been used successfully for
clinical analysis, particularly with cases that
have a low (or no) mitotic index.
● It is not useful for detecting balanced
rearrangements.

Slide 24. FISH panels for B cell disorders. Results from a

peripheral blood sample from a patient with CLL,
hybridized with the Abbott Molecular CLL probe panel with
addition of an MYB probe.

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 ● Whole chromosome probe
● Ratio-labeled probes are used to create a
distinct computer-generated false color for
each chromosome.
● Useful for complex rearrangements, such as
those seen in neoplastic disorders and solid
tumors.

Slide 27. The utility of metaphase CGH is illustrated by

the CGH profiles of a case with an insertion of unknown
material into the short arm of chromosome 4. The
chromosomal profiles reveal a gain of 15q (highlighted in
orange).

Slide 31. M-FISH of the pre-B ALL derived cell line RS4; 11
showing (A) blended colours generated by merging the
separated fluorochrome images, (B) pseudocolours
generated using the colour scheme in Fig. 1, and (C) colour
karyotype showing the t(4;11)(q21;a23), i(7q) and trisomy 8.
● each chromosome has individual color

MULTICOLOR BANDING (mBAND) ANALYSIS

● Uses chromosome-specific mixtures of partial

chromosome paints that are labeled with
various fluorochromes.
● colors different bands
● A computed program analyzes metaphase
chromosome data and produces a
pseudocolored, banded karyotype with an
estimated resolution of 550 bands, regardless
of chromosome length.
MULTIPLEX FISH (M-FISH) ● Advantageous for the determination of
breakpoints and the analysis of
● A technique that allows the investigator to intrachromosomal rearrangements and can be
view a karyotype so that each chromosome is particularly useful in preparations with shorter
“painted” with a different color. chromosomes.

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 Slide 35. Fiber-FISH analyzing the association of CL14
and CL34 repeats with telomeric DNA. (a) Five
representative fiber-FISH signals derived from probes
pAtT4 (red) and CL14 (green). (b) Five representative
fiber-FISH signals derived from probes pAtT4 (red) and
CL34 (green). Only the most proximal part of each
CL14/CL34 signal was included in each image. Note the
different sizes of the telomeric DNA and gap in the
junctions, indicating these signals may be derived from
different chromosomal ends. (c) Fiber-FISH analysis of
telomere (yellow), CL34 (red), and CL14 (green). The CL34
signals within the 4 images show significantly different
Slide 33. Multicolor banding. (a) Region-specific probes sizes. Only the most proximal part of each CL14 signal was
labeled with different partial chromosome patins (PCP) included in the image.
and computer false color (MetaSystems’ mBAND)
produces a definable number of colored bands per PRIMED IN SITU LABELING (PRINS)
chromosome, regardless of chromosome length. (b) This
example shows an abnormal X chromosome (right ● Essentially PCR on a slide.
homolog of each pair). ● Amplifying sequence is exponential
● Not homologous pair ● Primers of interest are hybridized on a slide
and then subjected to cycles of denaturation,
reannealing, and elongation that are used to
FIBER FISH
incorporate labeled nucleotides. The labels are
then detected fluorescently, or labeled
● A technique that is almost entirely used for
nucleotides are incorporated during the
research.
reaction.
● It allows the chromosomes to be stretched out
● Can differentiate hybridization with the alpha
and elongated.
satellite sequences for chromosomes 13 and
○ Stretched out = All sequences are exposed!
21, something that cannot be done with
● The probes are applied and can be physically
traditional FISH.
ordered on the fibers.
● This provides a much higher spatial resolution
and allows for correct orientation and
placement of probes and for precise mapping
of probes.

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 Slide 37. Metaphase chromosomes are subjected to Slide 39. Reverse FISH of a patient with an abnormal
PRINS with alpha satellite oligonucleotides specific for chromosome 8 (a) G-banding suggested a duplication of
chromosomes X, 11, and 17. Bright yellow fluorescein bands 8p23.1 p23.3. Two pairs of chromosomes 8 are
staining is seen at the centromeres of these shown; arrows indicate the additional band. This band
chromosomes. was microdissected, and the DNA was amplified, labeled,
and used as a FISH probe (b, c) Hybridization to normal
chromosomes.
REVERSE FISH

(b) The same metaphase is imaged with reverse DAPI to

● Used to identify material of unknown origin.
approximate G-banding patterns and identify the 2
● This unidentified material, such as marker
chromosomes 8, and with (c) typical DAPI staining. Arrows
chromosome or duplication, is flow sorted or
indicate both chromosomes 8 (d) Hybridization back to a
microdissected off a slide after G-banding. The
metaphase from the patient, demonstrating that 1
DNA from this material is extracted,
chromosomes 8 contains a duplication (arrow). The
PCR-amplified and labeled with a
reverse FISH results confirm the initial interpretation.
fluorochrome. This is then used as a probe and
hybridized to normal or patient metaphase
chromosomes to identify the origin of the *Under CGH but walang specified na topic or
unknown material. subtopic
● Copy-number variations (CNVs) are
alterations of the DNA of a genome that results
in the cell having an abnormal number of
copies of one or more sections of the DNA.
● Large regions of the genome have been deleted
(fewer than the normal number) or duplicated
(more than the normal number) on certain
chromosomes.
● Amplifications and deletions can contribute to
tumorigenesis.
● Amplification is the most common change
seen in malignancies.
● Detection and mapping provides an approach
to associate an aberration with a disease
phenotype and localizing critical genes -
Biomarkers.

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 ● Prognosis and therapeutics. ● CGH is based onco-hybridization of 2
● Resistance and susceptibility to disease. differentially labeled genomic DNAs (eg. tumor
○ Ex: HIV and SLE and normal) to human metaphase
● Mental retardation, developmental delay and chromosome spreads.
seizure disorders. ● It is based on the co-hybridization of
● Dysmorphic features and multiple congenital differentially labelled test and reference DNA
anomalies. onto metaphase spreads, which usually have
● Schizophrenia and autism spectrum disorder. been prepared from peripheral blood
lymphocytes of a healthy donor.
COMPARATIVE GENOMIC HYBRIDIZATION ● The signal intensity ratios of the 2 labels along
the chromosomes then reflect DNA copy
● Comparative Genomic Hybridization (CGH) or number changes in the test genome relative to
Chromosomal Microarray Analysis (CMA) is a the reference genome.
molecular-cytogenetic method for the analysis ● Resolution is limited to about 3 - 10 Mb.
of copy number changes (gains/losses) in the
DNA content of a given subject’s DNA and often
in tumor cells.
● First described in 1993 by Kallioniemi et. al.
● DNA from subject tissue and from normal
control tissue (reference) are each labeled with
different tags.
● Hybridized to metaphase chromosomes or, for
array- or matrix- CGH.
● Regional differences in the fluorescence ratio
of gains/losses vs. control DNA can be detected
and used for identifying abnormal regions in
the genome.
● CGH, a special FISH technique (dual probes), is
applied for detecting all genomic imbalances.
● The basics of technique is comparison of total
genomic DNA of the given sample DNA (e.g. Slide 14.
tumor DNA) with total genomic DNA of normal
cells.
● Typically, an identical amount of both tumor
and normal DNAs is labeled with 2 different
fluorescent dyes; the mixture is added and
hybridized to a normal lymphocyte metaphase
slide.
● A fluorescent microscope equipped with a CCD
camera and an image analysis systems are
used for evaluation.
● CGH is used to determine copy number
alterations of genome in cancer and those
cells whose karyotype is hard or impossible to Slide 15. After extraction of test DNA (i.e. from a tumor
prepare or analyze. sample) and normal DNA (i.e. from peripheral blood), the
● CGH will detect only unbalanced chromosomal samples are differentially labeled with discernable
changes. Structural chromosome aberrations fluorochromes (i.e. tumor DNA with FITC [green] and
such as balanced reciprocal translocations or control DNA with RITC [red]).
inversions can not be detected, as they do not
change the copy number.

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 ● The genomes are combined with an excess of ● A gain of chromosomal region in the test
cot-1 DNA and hybridized to metaphase sample would result in an increased intensity
chromosomes. of green fluorescence.
● Background hybridization due to repetitive ● A loss within a chromosomal region in the
DNA sequences is a common problem in tumor would be indicated by a shift towards
assays. red intensities.
● Cot-1 DNA blocking reagent blocks repetitive ● CGH analysis software measures fluorescence
DNA sequences and prevents nonspecific intensity values along the length of the
hybridization. chromosomes and translates the ratios into
chromosome profiles.
● The ratio of green to red fluorescence values is
used to quantitate genetic imbalances in
tumor samples.

● Images of metaphase spreads are then

acquired (charged coupled device) CCD camera
and fluorochrome-specific optical filter sets to
capture the FITC and TRITC fluorescence.
● Differences in fluorescence intensity values
between tumor and control DNA represent
gains and losses of specific chromosomes or
chromosomal regions.

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 ARRAY CGH: THE COMPLETE PROCESS

Step 1 - 3: Patient and control DNA are labeled with

fluorescent dyes and applied to the microarray.

Step 4: Patient and control DNA compete to attach, or

hybridize, to the microarray.

Step 5: The microarray scanner measures the

fluorescent signals.
Step 6: Compute software analyzes the data and
generates a plot.

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